Environmental and Experimental Botany 171 (2020) 103948

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Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

Leaf gas exchange and stable carbon isotope composition of redbay and T
avocado trees in response to laurel wilt or drought stress
Raiza Castillo-Argaeza, Bruce Schaffera,*, Aime Vazqueza,1, Leonel D.S.L. Sternbergb
a
Tropical Research and Education Center, University of Florida, 18905 S.W. 280 Street, Homestead, FL 33031, USA
b
Biological Sciences Department, University of Miami, 1320 South Dixie Highway, Coral Gables, FL 33146, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Symptoms of laurel wilt, a vascular plant disease caused by the fungus Raffaelea lauricola, are similar to those of
Lauraceae drought stress. We compared physiological responses of redbay and avocado trees infected with laurel wilt to
Raffaelea lauricola responses of trees with drought stress. Trees were either inoculated with R. lauricola, subjected to drought stress,
Net CO2 assimilation or irrigated daily and not inoculated (non-stressed, control). Disease symptoms, net CO2 assimilation (A), sto-
Stomatal conductance
matal conductance of water vapor (gs), substomatal CO2 concentration (Ci), intrinsic water use efficiency
Plant disease
(WUEi), leaf chlorophyll index (LCI), the ratio variable to maximum leaf chlorophyll fluorescence (Fv/Fm), leaf
nitrogen (N) concentration, and the ratio of 13C to 12C (δ13C) in leaves were determined prior to inoculation and
the imposition of drought stress and when plants showed moderate to severe stress symptoms. Laurel wilt-
infected and drought-stressed trees of each species had lower A and gs and higher Ci, Fv/Fm, and LCI than non-
stressed trees. Redbay trees with laurel wilt had significantly higher (less negative) leaf δ13C values than non-
stressed trees, whereas no significant differences were found in leaf δ13C values among avocado treatments. In
avocado trees with laurel wilt, there was a negative linear correlation between δ13C values and Ci but no sig-
nificant correlation between δ13C values and leaf N in the inoculated trees. Inhibition of photosynthesis in
avocado and redbay infected with laurel wilt was determined to be a result of the both stomatal and biochemical
factors.

1. Introduction
Laurel wilt is a vascular disease caused by the fungus Raffaelea
lauricola T. C. Harr., Fraedrich & Ahayeva (Ophistomatales:
Ophiostomataceae), which infects most members of the Lauraceae fa-
mily, including avocado (Persea americana Mill.) and redbay (Persea
borbonia L.). The disease was first observed to be spread by the redbay
ambrosia beetle, Xyleborous glabratus Eichhoff (Coleoptera:
Curculionidae: Scolytinae), but several additional species of ambrosia
beetles have now been shown to be vectors of the disease (Carrillo
et al., 2012, 2014; Ploetz et al., 2017). Both the fungus and the redbay
ambrosia beetle were accidently introduced into the United States 14
years ago. Since that time, an estimated half billion native trees in the
southeastern United States and more than 7000 avocado trees in
southern Florida have been destroyed by the disease (Kendra et al.,
2013; Mosquera et al., 2015).
Several tree responses to vascular wilt disease are similar to re-
sponses to drought stress such as reduced stomatal conductance (gs),
transpiration (E), and wilting (Li et al., 2016; Ploetz et al., 2015;
Pouzoulet et al., 2014; Urban and Dvořák, 2014). Invasion of the vas-
cular system by a fungal pathogen elicits the formation of gums and
gels, resulting from the dissolution of xylem cell walls and the forma-
tion of tyloses, which are outgrowths of xylem vessel cells (Beckman,
1971; De Micco et al., 2016). These defense responses wall off the pa-
thogen, preventing it from spreading throughout the plant. In elm trees
with Dutch elm disease caused by the fungus Ophiostoma novo-ulmi, the
blockage of the xylem vessels resulted in reduced water transport
(Roberts, 1972; Li et al., 2016) and decreased gs, E, and net CO2 as-
similation (A) in infected trees (Urban and Dvořák, 2014; Pouzoulet
et al., 2014).
As with other vascular wilt diseases, laurel wilt causes the formation
tyloses and gels and gums that occlude the xylem vessels (Inch et al.,
2012; Inch and Ploetz, 2012), causing a reduction in stem hydraulic
conductivity, sap flow (Inch and Ploetz, 2012; Ploetz et al., 2015;
Dreaden et al., 2017), gs, E, and A (Ploetz et al., 2015). Eventually, leaf
wilting and necrosis occur (Fraedrich et al., 2007). Compared to other

⁎
Corresponding author.
E-mail address: bas56@ufl.edu (B. Schaffer).
1
Current address: USDA, ARS, Subtropical Horticulture Research Station, 13601 Old Cutler Road, Miami, FL, 33158, USA.

https://doi.org/10.1016/j.envexpbot.2019.103948
Received 15 July 2019; Received in revised form 26 October 2019; Accepted 10 November 2019
Available online 11 November 2019
0098-8472/ © 2019 Elsevier B.V. All rights reserved.
R. Castillo-Argaez, et al.
vascular fungal tree pathogens, R. lauricola spreads so rapidly
throughout the xylem that there is an exponential development of ty-
loses, and instead of walling off the disease, the entire vascular system
quickly becomes blocked (Inch et al., 2012). This happens so rapidly
that mature trees with laurel wilt generally die within months after
contracting the disease (Ploetz et al., 2012). Thus, tree responses to
laurel wilt are similar to those of severe drought stress.
Similar to laurel wilt, drought stress results in stomata closing to
prevent water loss, thereby reducing gs, E, and A (McDowell et al.,
2008; Flexas and Medrano, 2002). Also, like laurel wilt, severe drought
stress impairs plant metabolism and water transport and leads to
wilting, plant desiccation and death (Tezara et al., 1999; Lawlor and
Cornic, 2002). In mature avocado trees, mild to moderate water stress
(stem water potential of -0.4 MPa) decreased gs thereby reducing A, and
severe water stress (stem water potential -1.0 MPa) caused stomata to
close (Sterne et al., 1977; Sharon, 1999).
Carbon (C) isotope discrimination during photosynthesis, reflected
in the isotope composition in plants, has been a reliable tool to differ-
entiate between plant metabolic pathways and for selection of cultivars
with high water use efficiency (Bender, 1971; Osmond, 1978; Hall
et al., 1993; Cerling et al., 1999; Crayn et al., 2004; Condon et al.,
2004). The processes that affect the 13C/12C ratio (δ13C) in plants are
those that can fractionate carbon isotopes, the diffusion of 13C and 12C
in air, and enzymatic fractionation of C (O’Leary, 1981, 1993; Farquhar
et al., 1982). These processes discriminate against the heavier 13C iso-
tope. During drought stress the diffusion of CO2 into the plant is af-
fected by stomatal closure, which increases the leaf carbon isotope
composition and decreases carbon isotope discrimination (Farquhar
et al., 1989).
The objective of this study was to determine if the mechanism(s) for
reductions in A in laurel wilt-infected avocado and redbay trees are
stomatal (due to closing of stomata, similar to a water stress response),
biochemical (i.e., inhibition of carbon fixation), or a combination of
both factors.
2. Materials and methods
2.1. Plant material and experimental design
From 13 September to 14 October 2018, two-year-old redbay (P.
borbonia) and avocado (P. americana cv. Russell) trees (with a height of
116.1 ± 2.6 cm and diameter of 16.5 ± 0.2 mm; mean + std. error)
were obtained from commercial plant nurseries and transplanted into
18.9-L plastic pots filled with a commercial potting medium (ProMix®,
Premier Tech, Quebec, Canada). Trees were then placed in a controlled-
temperature greenhouse at the University of Florida, Tropical Research
and Education Center in Homestead, Florida. Temperature and relative
humidity inside the greenhouse were recorded with a HOBO Pro v2
datalogger (Onset Computer Corp., Bourne, Massachusetts, USA) lo-
cated about 1 m above the tree canopies. During the experiment, daily
air temperature in the greenhouse ranged from 21.5 °C to 32.7 °C with a
mean of 26.1 ± 0.3 °C and relative humidity ranged from 42.3% to
96.2% with a mean of 77.8 ± 2.9 %. Light intensity (photosynthetic
photon flux; PPF) inside the greenhouse on a cloudless day was 60 % of
ambient PPF as determined with a quantum sensor (LI-185, Li-Cor
Instruments, Inc., Lincoln, Nebraska, USA) connected to a data-logger
(LI-1000, Li-Cor Instruments, Inc., Lincoln, Nebraska, USA).
Three treatments were established: 1) drought; the amount of irri-
gation was decreased over time to simulate the progressive stress
symptoms from laurel wilt disease, 2) laurel wilt; trees inoculated with
R. lauricola and irrigated daily, and 3) control, trees irrigated daily. The
experiment was arranged in a completely randomized design with six
single-tree replicates in each treatment for each tree species.
2
Environmental and Experimental Botany 171 (2020) 103948
2.2. Soil water content in the drought treatment
Prior to beginning the experiment, plants in the drought treatment
were irrigated and allowed to drain to field capacity. Each plant (in-
cluding the potting medium and pot) was then weighed. Soil moisture
content in each pot was determined with a SM150 T soil moisture
sensor connected to a HH150 voltage meter (Delta-T devices Ltd,
Cambridge, UK). Soil moisture content (θ) was calculated as:
θ = [ ε − α 0/ a1] (1)
where
ε = 1.0 + 14.4396V − 31.2587V 2 + 49.0575V 3 − 36.5575V 4
+ 10.7117V 5 (2)
V is the sensor output in volts, and α1 and α0 are the soil calibration
values of 1.16 and 7.09, respectively.
For daily monitoring of soil water tension, a tensiometer (Irrometer,
Inc., Riverside, California, USA) was installed in the potting medium of
each plant with the tip placed 20 cm deep into the medium about half-
way between the plant stem and the side of the pot. Each plant was
weighed daily and the quantity (g) of water lost the previous day was
added to the potting medium to maintain the soil water content at a
specific, pre-determined soil water tension. Since the purpose of the
drought treatment was to simulated laurel wilt symptom progression,
irrigation was not completely cut off from plants because this could
have caused a sudden decline of soil moisture content and soil water
tension. Instead trees in the drought treatment were shifted through 3
stages of water availability: During the first 11 days of the experiment,
the potting medium was maintained at a soil tension of ∼14 kPa, which
provides adequate soil moisture and prevent water stress of avocado
(Schaffer et al., 2013). From days 12–24, soil tension was maintained at
∼40 kPa (moderate water stress) and from day 25 to the end of the
experiment, soil tension was maintained at ∼71 kPa (severe water
stress). Tensiometers were also placed in three pots (replicates) each of
the control and laurel wilt treatments to be sure that that soil water
tension in those treatments was maintained at not less than ∼14 kPa
(30 % of soil moister content) throughout the experiment.
2.3. Plant inoculation
Four holes, 2 mm diameter and 4 mm deep, were drilled into each
tree at a 45° angle at equidistant locations around the main stem about
20 cm above the soil surface. For trees in the laurel wilt treatment, a 25-
μl liquid suspension culture of R. lauricola conidia (Isolate RL4, spe-
cimen CBS 127349; deposited at the CBS Fungal Biodiversity Center,
Utrecht, the Netherlands) was pipetted into each hole. Each tree was
inoculated with a total of 3,000 conidia (Ploetz et al., 2012, 2015).
Trees in the laurel wilt treatment were irrigated daily.
Holes were also drilled as previously described into trees in the
drought and control treatments, to account for possible wounding ef-
fects from the inoculation procedure in the laurel wilt treatment, and
25 μm of deionized water was pipetted into each hole.
2.4. Leaf gas exchange, chlorophyll index and chlorophyll fluorescence
Leaf gas exchange variables [A, gs, and substomatal CO2 con-
centration (Ci)] were measured for each replicate in each treatment
with a CIRAS-3 portable gas analyzer (PP systems, Amesbury,
Massachusetts, USA). Intrinsic water use efficiency (WUEi) was calcu-
lated as A/E. The PPF in the leaf cuvette was set at 1000 μmol m-2 s-1
and the CO2 concentration of air entering the cuvette was set at
410 μmol mol-1. The flow rate of air into the leaf cuvette was set at
300 ml min-1 and VPD of the reference air entering the cuvette was 1.34
Kpa. Leaf gas exchange measurements were made on one fully ex-
panded, recently matured leaf (Schaffer et al., 1991) of each tree
R. Castillo-Argaez, et al. Environmental and Experimental Botany 171 (2020) 103948

Table 1
Soil matric potential and soil water content of avocado and redbay trees in the control, drought, and laurel wilt treatments at three days after inoculation (DAI).
Soil matric potential (kPa) Soil moisture content (%)

Control Drought Laurel wilt Control Drought Laurel wilt

DAI -1 to 10 4.87 ± 0.57b 14.25 ± 1.55a 5.87 ± 0.28b 49.40 ± 0.04a 30.00 ± 0.01b 47.18 ± 0.03a
DAI 11 to 23 3.31 ± 0.56b 38.41 ± 9.7a 4.31 ± 0.77b 50.81 ± 0.03a 16.44 ± 0.05b 52.54 ± 0.02a
DAI 24 to 31 2.93 ± 0.64b 71.40 ± 7.30a 2.25 ± 0.65b 52.26 ± 0.04a 2.44 ± 0.01b 49.51 ± 0.01a

Data are means combined for both species for a total of 12 replicates per drought treatment and 6 replicates for control and laurel wilt treatments. Different letters
within rows indicate significant differences (P ≤ 0.05) among treatments according to a Waller-Duncan K-ratio test.

maximum chlorophyll fluorescence; Fv/Fm) was recorded. Measure-

ments were made prior to treatment initiation (day -1), on the in-
oculation day (day 0), and then three times per week (Monday, Wed-
nesday and Friday) until the end of the experiment.

2.5. Carbon isotope analysis

To ensure taking into account the treatment effects during the de-
velopment of sampled leaves, only young leaves that emerged and ex-
panded fully after treatments began were collected for carbon isotope
analysis at -1, 21, 25 and 31 DAI. Leaves were cleaned with Kimwipes®
to remove any dust, petioles were removed, and leaves were dried for
72 h at 50 °C in a drying oven. Leaf samples were cut into small pieces,
placed in 2.0 mm polymer micro-centrifuge tubes with two 5 mm dia-
meter stainless steel balls per tube, and ground to fine powder using a
Retsch® mixer mill MM 200 at a frequency of 25 s-1. Three to four mg of
each sample were placed in a tin elemental micro-analysis capsule and
rolled into a small ball. The concentrations of 13C and 12C in each
sample was determined with a Pyrocube elemental analyzer connected
to an Isoprime isotope ratio mass spectrometer (Elementar, Hanau,
Germany). The carbon isotope ratio for each sample was expressed as:
δ 13 C = [(Rsample /Rstandard ) − 1]*1000 (3)
13 12
where Rsample, and Rstandard are the molar ratio C/ C of the sample
and of a Belemnita americana fossil carbonate standard from the Pee Dee
geological formation in South Carolina (PDB), respectively. The preci-
sion of isotope measurements was ± 0.1‰. The total percentage of C
and nitrogen (N) in each leaf was determined with the mass spectro-
meter at the same time as carbon isotope determinations.

2.6. Plant symptom and disease assessment

Fig. 1. Progression of external symptoms based on a visual scale (1 = no
symptoms, 10 = 100 % of the canopy exhibiting symptoms) in redbay and
avocado trees in control, drought, and laurel wilt treatments. Different letters
indicate significant differences among treatments (P < 0.05) according to a
repeated measures ANOVA. Values are means ± SE.
between 900 and 1200 h. On the same days that leaf gas exchange was
measured, the leaf chlorophyll index (LCI) was measured on the adaxial
side of three fully expanded, recently matured leaves of each plant with
a SPAD meter (model 502, Minolta, Inc., Osaka, Japan). Also, on the
same days, chlorophyll fluorescence was measured on two fully ex-
panded, recently matured leaves per plant with a portable fluorescence
meter (model OS-30p, Opti-Sciences Inc., Hudson, NH, USA). Leaves
were acclimated in the dark for 30 min prior to measurements. The
maximum quantum efficiency of Photosystem II (the ratio of variable to
Water stress and disease symptom severity were assessed daily for
plants in each treatment using a visual rating scale based on the per-
centage of canopy exhibiting leaf wilting, necrosis and stem dieback
symptoms, where: 1=no symptoms, 2 = 1–11%, 3 = 12–23%,
4 = 24–34%, 5 = 35–47%, 6 = 48–59, 7 = 60–71%, 8 = 72–87%,
9 = 88–99% and 10 = 100 % (Ploetz et al., 2012).
At the end of the experiment, internal sapwood discoloration (vas-
cular staining) was assessed for plants in each treatment using the same
visual scale (i.e. 1 = no discoloration, 10 = 100 % of the sapwood
discolored). Three stem segments (2 cm in length) were collected at
2 cm and 30 cm above the inoculation point and at the stem tip of all
trees in each treatment and plated on a semi-selective medium
(CSMA+) for recovery R. lauricola (Ploetz et al., 2012).
2.7. Data analyses
Data from redbay and avocado trees were statistically analyzed se-
parately because we were not interested in species comparisons.
Statistical differences among treatments for soil water content were
analyzed by one-way analysis of variance (ANOVA) and a Duncan-
Waller K-ratio test. Leaf gas exchange variables, leaf chlorophyll index,
and chlorophyll fluorescence were analyzed by repeated measurements

3
R. Castillo-Argaez, et al.
Fig. 2. Net CO2 assimilation (A) of redbay and avocado trees in control,
drought, and laurel wilt treatments. Different letters indicate significant dif-
ferences among treatments (P < 0.05) according to a repeated measures
ANOVA. Values are means ± SE.
ANOVA. The relationship between A and gs was determined by linear
regression analysis and the relationships among δ13C, gs, and N were
determined by stepwise linear regression. Differences in δ13C among
treatments were determined by a one-way ANOVA and a test for least
significant difference (LSD). When a linear relationship was observed
prior to treatment initiation between N and δ13C, N was used as a
covariate to analyze δ13C differences among treatments. Differences in
external symptoms severity among treatments were determined by a
repeated measures ANOVA after transforming the data using a natural
logarithm transformation to normalize the distribution. Internal
symptom development (sapwood discoloration) was analyzed with a
non-parametric Wilcoxon analysis followed by a Bonferroni correction.
All statistical analyses were done with SAS statistical software (version
9.4, SAS Institute, Cary, North Carolina, USA).
3. Results
Plants in the drought treatment exhibited progressive water stress
over time, with a higher soil matric potential (71.4 kPa) and lower soil
water content (2.4 %) than the control or laurel wilt treatments, which
each had a soil matric potential between 2–3 kPa and soil water content
of ∼50 % by 24–31 DAI (Table 1).
Symptom development differed among treatments in each species
4
Environmental and Experimental Botany 171 (2020) 103948
Fig. 3. Stomatal conductance (gs) of redbay and avocado trees in control,
drought, and laurel wilt treatments. Different letters indicate significant dif-
ferences among treatments (P < 0.05) according to a repeated measures
ANOVA. Values are means ± SE.
(Fig. 1). In general, trees in the drought treatment first exhibited leaf
epinasty and loss of leaf turgor at 18 DAI and necrotic leaves were
observed at 21 DAI. In contrast, leaves of trees in the laurel wilt
treatment did not wilt but became necrotic beginning at 13 DAI. For
both species, trees in the control treatment did not exhibit signs of
wilting or necrosis during the experiment, thus the average external
symptom rating was 1.
There was a significant statistical interaction between treatment and
DAI for redbay trees (F = 52.1; df = 2,32; P < .0001). At the end of
the experiment, redbay trees in the drought treatment had fewer
symptoms than trees in laurel wilt treatment, 5.2 ± 0.5 and
7.74 ± 0.5 (mean ± std. error), respectively. A significant interaction
was observed between treatment and sampling date for avocado trees
(F = 26.3; df = 2,32; P < .0001). After 31 DAI, avocado trees in the
drought and laurel wilt treatments had similar external symptom de-
velopment, with external symptom ratings of 8.1 ± 0.6 and
7.5 ± 0.9, respectively compared to the control treatment with an
average symptom rating 1 (Fig. 1). Internal symptoms (sapwood dis-
coloration) was only observed in trees in the laurel wilt treatment and
not in any trees in the drought or control treatments. Redbay trees in
the laurel wilt treatment had an internal symptom rating of 7.3 ± 1.1,
significantly higher than that of the control and drought treatments
(Z = 21, P = 0.01), which each had a rating of 1. Avocado trees in the
R. Castillo-Argaez, et al.
Fig. 4. Intrinsic water use efficiency (WUEi) of redbay and avocado trees in
control, drought, and laurel wilt treatments. Different letters indicate sig-
nificant differences among treatments (P < 0.05) according to a repeated
measures ANOVA. Values are means ± SE.
laurel wilt treatment had an internal discoloration rating of 8.5 ± 0.5,
which was significantly higher than that of the control or drought
treatments (Z = 21, P = 0.01). There were no significant differences in
internal disease rating between the control and drought treatments
(Z = 39, P = 1.0). At the end of the experiment, R. lauricola was re-
covered from all stem sections of all plants in the laurel wilt treatment
but not from any trees in the other two treatments for each species.
In both species, A, gs, and WUEi decreased over time in the drought
and laurel wilt treatments (Figs. 2–4) and were lower than in the
control treatment. The Ci was significantly higher in the drought and
laurel wilt treatments than in the control treatment from 19 DAI to the
end of the experiment in both species (Fig. 5). In general, the LCI de-
creased more rapidly over time in the laurel wilt treatment than in
drought treatment (Fig. 6). However, at the end of the experiment the
LCI was not significantly different between the drought and laurel wilt
treatments for either species. The Fv/Fm was higher in the control
treatment than in the drought or laurel wilt treatments for both species,
and the rate of decline of the Fv/Fm over time was greater for trees in
the laurel wilt treatment than in the drought treatment (Fig. 7).
For redbay trees, there was a significant linear relationship between
A and gs for trees in the control (P < .0001, R2 = 0.76), drought
(P < .0001, R2 = 0.85), and laurel wilt (P < .0001, R2 = 0.86)
5
Environmental and Experimental Botany 171 (2020) 103948
Fig. 5. Substomatal CO2 concentration (Ci) of redbay and avocado trees in
control, drought, and laurel wilt treatments. Different letters indicate sig-
nificant differences among treatments (P < 0.05) according to a repeated
measures ANOVA. Values are means ± SE.
treatments (Fig. 8). Similarly, for avocado trees there was a positive
linear relationship between A and gs for trees in the control
(P < .0001, R2 = 0.45), drought (P < .0001, R2 = 0.89), and laurel
wilt (P < .0001; R2 = 0.88) treatments (Fig. 8). For both redbay and
avocado trees in the drought and laurel wilt treatments, gs and A were
very low for all trees (replicates) after 11 DAI, with values of 0 and/or
negative values at the end of the experiment, an indication that all trees
in these treatments were very stressed. In avocado, there was a sig-
nificant effect of treatment on the leaf N content (F = 3.44; df = 2,48;
P = .04), which was higher in the control than in the drought treat-
ment, whereas there was no significant difference in leaf N content
among redbay treatments (F = 1.36; df = 2,47; P = 0.26) (Table 2).
Also, leaf C content in avocado trees was significantly higher (F = 5.0;
df = 4,48; P = 0.01) in the drought treatment than in the control
treatment, but there was no significant difference between the laurel
wilt and the control treatments (Table 2). In contrast, redbay trees in
the laurel wilt treatment had a higher leaf C content than trees in the
control treatment (F = 3.42; df = 2,47; P = 0.04). Also, redbay trees in
the laurel wilt treatment had significantly higher (less negative) leaf
δ13C than trees in the control treatment (F = 6.3; df = 2,46;
P = 0.001), whereas no significant differences were found in leaf δ13C
among avocado treatments (F = 1.2; df = 2,48; P = 0.30). In avocado
trees, there was a linear relationship between δ13C and Ci in the laurel
R. Castillo-Argaez, et al.
Fig. 6. Leaf chlorophyll index (LCI) of redbay and avocado trees in control,
drought, and laurel wilt treatments. Different letters indicate significant dif-
ferences among treatments (P < 0.05) according to a repeated measures
ANOVA. Values are means ± SE.
wilt treatment (P = 0.0006, R2 = 0.74), whereas there was no sig-
nificant relationship between these variables for trees in the control
treatment (P = 0.85) or drought treatment (P = 0.95) (Fig. 9). In
redbay trees, there was no significant relationship between δ13C and Ci
in the control (P = 0.10), drought (P = 0.82), or laurel wilt (P = 0.37)
treatments.
In redbay, there was a significant linear relationship between δ13C
and leaf N content (P = 0.0001, R2 = 0.54), in the control treatment,
but no significant relationship between these variables in the laurel wilt
or drought treatments (Fig. 10). For avocado, there was a significant
linear relationship between δ13 C and leaf N content in control
(P < .0001, R2 = 0.75), drought (P = 0.005, R2 = 0.40), and laurel
wilt (P = 0.01, R2 = 0.50) treatments (Fig. 10).
4. Discussion
For redbay and avocado, trees in the drought and laurel wilt
treatments displayed similar symptoms such as leaf wilting and ne-
crosis. However, symptom progression differed among those two
treatments. After inoculating trees with conidia of R. lauricola, leaves
did not show a gradual loss of turgor, but suddenly turned reddish
brown and exhibited complete loss of turgor approximately two weeks
6
Environmental and Experimental Botany 171 (2020) 103948
Fig. 7. The ratio of variable to maximum chlorophyll fluorescence (Fv/Fm) of
redbay and avocado trees in control, drought, and laurel wilt treatments.
Different letters indicate significant differences among treatments (P < 0.05)
according to a repeated measures ANOVA. Values are means ± SE.
after inoculation. In contrast, leaves of trees in the drought treatment
exhibited a progressive loss of turgor followed by curling, and then
necrosis which occurred three to four weeks after treatments began.
The rapid development of leaf necrosis in trees with laurel wilt is a
common response in species that are very susceptible to the disease
including redbay (Fraedrich et al., 2007) and avocado trees (Ploetz
et al., 2012). Thus, the more rapid development of leaf necrosis in trees
with laurel wilt compared to those with drought stress is presumably
indicative of the more sudden inhibition of water transport in trees with
laurel wilt compared to trees stressed solely by reducing water avail-
ability. Despite the attempt to progressively impose tree stress at about
the same rate in the drought treatment as in laurel wilt treatment, it
proved very difficult to impose an abiotic stress at the same rate as a
biotic stress.
An initial response of trees to either drought stress or invasion by a
root or vascular pathogen is stomatal closure to avoid or reduce water
loss, which results in reduced gs and A (Barrientos-Priego and
Rodríguez-Ontiveros, 1994; Chartzoulakis et al., 2002; Li et al., 2016;
Ploetz et al., 2015; Schaffer and Ploetz, 1989). In the present study, the
rates of decline in A, gs, and WUEi were greater in the laurel wilt
treatment than in the drought treatment. Ploetz et al. (2015) also ob-
served an increase of Ci in avocado trees infected with laurel wilt. An
R. Castillo-Argaez, et al. Environmental and Experimental Botany 171 (2020) 103948

Fig. 8. Relationship between net CO2 assimilation (A) and stomatal conductance (gs) of redbay and avocado trees in the control, drought, and laurel wilt treatments.
The linear regression line is shown.

Table 2
Total nitrogen (N), carbon (C), and carbon isotope composition (δ13C) of avocado and redbay leaves in the control, drought, and laurel wilt treatments.
Avocado Redbay

Variable Control Drought Laurel wilt Control Drought Laurel wilt

N (%) 1.83 ± 0.13a 1.36 ± 0.13b 1.57 ± 0.11ab 2.12 ± 0.1a 2.18 ± 0.08a 1.90 ± 0.07a
C (%) 45.66 ± 0.72b 48.901 ± 0.85a 47.58 ± 1.11ab 46.52 ± 0.80b 48.69 ± 0.94ab 49.80 ± 0.98a
δ13C −30.34 ± 0.24a −30.18 ± 0.24a −30.22 ± 0.30a −29.5 ± 0.29b −29.41 ± 0.26ab −28.56 ± 0.18a

Different letters within rows indicate significant differences (P ≤ 0.05) among treatment of the same species according to a Waller-Duncan K-ratio test. N was used as
a covariate to analyze δ13C differences among treatments.

increase in WUEi due to decreased gs is a common response of plants to
mild or moderate water stress (Monclus et al., 2006; Tambussi et al.,
2007; Jaleel et al., 2008). However, in the present study, WUEi was
lower in the drought and laurel wilt treatments than in the controls, a
result of the stomatal closure and a decline in gs and A which negatively
affected WUEi. Prolonged or severe drought can inhibit carboxylation
and impair biochemical carbon fixation (Flexas and Medrano, 2002).
Severe drought also affects the activity of rubisco and ATP synthesis
(Tezara et al., 1999; Lawlor and Cornic, 2002) and electron transport as
noted by the reduction of Fv/Fm (Flexas et al., 1999), an indication of
damage to Photosystem II (Long et al., 1994). Thus, the inhibition of
biochemical C fixation results in an increase in Ci (Picon et al., 1996;

7
R. Castillo-Argaez, et al.
Fig. 9. Relationship between carbon isotope composition (δ13C) and sub-
stomatal CO2 concentration (Ci) of avocado leaves in the control, drought, and
laurel wilt treatments. Symbols represent data collected at -1, 21, 25 and 31
DAI. The regression line is also shown.
Monneveux et al., 2006).
In redbay and avocado trees, the LCI generally decreased in the
laurel wilt treatment prior to decreasing in the drought treatment, and
this occurred days after the reduction in leaf gas exchange variables.
Reduction of leaf chlorophyll content and Fv/Fm have been reported in
plants affected by fungal pathogens, such as elm trees with Dutch elm
disease, which was related to leaf senescence (Santos et al., 2005;
Oliveira et al., 2012). Leaf N content was slightly lower in redbay and
8
Environmental and Experimental Botany 171 (2020) 103948
avocado trees in the drought and laurel wilt treatments than in the
control treatment, suggesting a degradation of chlorophyll which re-
sulted in the reduced LCI.
In a previous study comparing Phytophthora root rot, caused by
Phytophthora cinnamomi, with drought stress of oak (Quercus ilex) trees,
Ruiz Gómez et al. (2018) observed differences in physiological response
to drought versus plant disease. Trees inoculated with P. cinnamomi
exhibited an initial decrease in A but recovered after the first infection
cycle, whereas in trees that were severely drought stressed, not only
was A inhibited but there was a decrease in photosynthetic efficiency as
determined by a non-recoverable decrease of Fv/Fm. In the present
study, Fv/Fm in redbay and avocado trees decreased rapidly over time
in both the drought and laurel wilt treatments. The difference in re-
sponse of Fv/Fm to laurel wilt than to Phytophthora root rot pre-
sumably was due to the more rapid decline in plants exposed to the
extremely aggressive laurel pathogen compared to those exposed to
Phytophthora root rot. For example, in avocado trees infected with P.
cinnamomi, some cultivars can regenerate roots to cope with the in-
fection (Kellam and Coffey, 1985), whereas laurel wilt appears to al-
ways be lethal in susceptible trees such as redbay and avocado
(Fraedrich et al., 2007; Ploetz et al., 2012).
An increase of 13C in plants exposed to drought stress has been re-
ported in several species (Dawson, 1993). However, in the present
study, trees in the drought treatment were exposed relatively quickly to
severe water stress to try to simulate the rapid desiccation due to laurel
wilt. This may explain why no significant differences in the 13C com-
position between trees in the control and drought treatments were
observed. Increased Ci in trees in the laurel wilt treatment indicated an
inhibition of CO2 fixation which presumably resulted in the disruption
of carbon isotope discrimination. However, there was no change in δ13C
in drought stressed trees compared to trees in the control treatment.
The relatively rapid onset of drought stress in the present study may
have resulted in little carbon fixation under dry conditions. The young
leaves used for carbon isotope analysis may have been synthesized from
pre-treatment carbon reserves and thus did not show the isotopic sig-
nature generally observed for trees under slight or moderate drought
stress. When comparing drought stress to Dutch elm disease, Roberts
(1972) suggested that the decreases in A in diseased trees is related to
biochemical factors or to pathogen-host signaling which can elicit the
production of phytotoxins.
There have been few reports of the effects of vascular wilt disease on
carbon isotope discrimination or composition. Daugherty et al. (2010)
found that δ13C increased in alfalfa plants as a result of infection by
Xylella fastidiosa, a xylem limited bacterium. The degree of carbon
composition in X. fastidiosa-infected alfalfa was also related to the strain
of the pathogen; plants infected with more pathogenic strains had
higher δ13C than those infected with less pathogenic strains. The dif-
ference between our results and those of Daugherty et al. (2010) could
be related to the differences in the disease progression and severity.
Alfalfa plants infected with X. fastidiosa can survive for several months
(Long et al., 2019), whereas trees highly susceptible to laurel wilt often
die within a couple of weeks or one month after inoculation (Fraedrich
et al., 2011; Ploetz et al., 2012; Spiegel and Leege, 2013).
In summary, in redbay and avocado trees, laurel wilt and drought
stress elicit similar responses such as impaired leaf gas exchange and
external stress symptoms such as leaf epinasty and necrosis. Our results
suggest that decreased A in redbay and avocado trees with moderate to
severe laurel wilt infection is not only due to reduced water transport
and reduced gs as a result of tyloses formation in the xylem, but plant
metabolism is also negatively impacted.
Author statement
The roles of each author in this work were as follows:
Raiza Castillo-Argaez, Bruce Schaffer, Leonel Sternberg:
Conceptualization
R. Castillo-Argaez, et al. Environmental and Experimental Botany 171 (2020) 103948

Fig. 10. Relationship between the carbon isotope composition (δ13C) and nitrogen (N) content in redbay and avocado leaves in the control, drought, and laurel wilt
treatments. Symbols represent data collected at -1, 21, 25 and 31 DAI. The regression line is shown.

Raiza Castillo-Argaez, Bruce Schaffer, Leonel Sternberg, Aime References

Vasquez: Methodology development and data collection
Raiza Castillo-Argaez, Bruce Schaffer, Leonel Sternberg: Data ana-
lyses and interpretation
Raiza Castillo-Argaez, Bruce Schaffer, Leonel Sternberg: Writing,
reviewing, editing, revising
Declaration of Competing Interest
None.
Acknowledgements
We thank Joshua Konkol, Randy Fernandez and Ana Vargas for
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